ONLY CORE PROTEIN
The adhesion of cells to the matrix is mainly mediated by the PG core protein. For example, the extracellular domain of the CD44 core protein binds different matrix component, like hyaluronan, collagens type I and II, laminin, fibronectin and osteopontin [Hardingham and Fosang, 1992; Hertweck et al., 2011; Louderbough and Schroeder, 2011]. The N-terminal domain of core protein is involved in hyaluronan binding giving it a different binding affinity for hyaluronan. The different glycosylation of the core protein and the molecular weight of Ha, in particular during inflammation seem to be implicated in the variation of the hyaluronan-binding affinity [Hardingham and Fosang, 1992]. In particular, while low molecular weight Ha stimulates cell growth, high molecular weight Ha inhibits proliferation [Hertweck et al., 2011]. This binding activates several pathway involved in cell proliferation, adhesion and migration. More precisely, it activates Rho and Rac1 GTPase that cause a reorganization of the actin cytoskeleton, ErbB2 tyrosine kinases, which lead to cell proliferation, and nuclear factot NFkB [Wu et al., 2005]. Osteopontin selectively binds to CD44 isoforms v6 and v7 and triggers signaling that promotes cell survival, migration and invasion and angiogenesis.
Versican binds hyaluronan molecules via its N-terminal globular domain and lectins via the C terminal region and it may also interact with fibronectin, tenascin R, fibulin 1 and 2, fibrillin 1and collagen type I reducing cell adhesion [Iozzo and Murdoch, 1996; Iozzo, 1998; Wight, 2002; Wu et
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al., 2005; Ricciardelli et al., 2009]. The versican C-terminal G3 domain is involved in the binding of β1 i ntegrins increasing phosphorylation of FAK and reducing H2O2-induced apoptosis [Wu et al., 2005]. The increased FAK phosphorilation causes an increased cell adhesion and spreading and a reduced migratory capacity [Wu et al., 2005]. In vitro experiments on NIH-3T3 fibroblasts and canecr cells show that soluble versican may reduce prostate cancer and melanoma cells adhesion to fibronectin-coated surfaces, promoting cancer cell proliferation and motility [Theocharis et al., 2010].
Also aggrecan, neurocan and brevican bind hyaluronan via their N-terminal globular domain I, in particular the G1 region, that binds lectins via the C-terminal region [Hardingham and Fosang, 1992; Iozzo and Murdoch, 1996; Iozzo, 1998]. Brevican upregulation and proteolytic cleavage affect cancer cell adhesion and promote cancer cell motility and its core protein is also involved in EGFR activation that determins an increasing in cell-adhesion molecules expression and secrection of fibronectin fibrils on the cell surface [Theocharis et al., 2010].
Perlecan may have different functions in the cell-matrix adhesion on the basis of the type of cell involved. In fact cells like endothelial cells or chondrocytes are urged by perlecan expression to adhere to the ECM while other cell types like that of the hematopoietic system may be rejected by the presence of the perlecan [Iozzo and Murdoch, 1996]. Some adhesive properties described for perlecan can be attributed to N-terminal domain [Iozzo and Murdoch, 1996]. For example perlecan N-terminal SEA module and the SGD tripeptides may bind laminin 111, collagen type IV, fibronectin, thrombomodulin, fibrillin 1 and PRELP [Bix and Iozzo, 2008]. The domain II contain the LDL attachment sites and the domain III, that is present in mouse but not conserved in human, contain an RGDS region that binds FGF7 and FGF-BP [Bix and Iozzo, 2008], whereas the domain IV of perlecan core protein is involved in neural cell adhesion via contact with N-CAM molecules [Iozzo, 1998] and it also binds nidogens, fibuli 2, fibronectin and collagen type IV [Bix and Iozzo, 2008]. The C-terminal domain V contains EGF repeats and binds nidogen 1, fibulin 2, β1 integrin, α2 integrin, FGF7, collagen XVIII, PRELP and α-dystroglycan [Bix and Iozzo, 2008]. To support the core protein functions, also GAG chains of perlecan are involved with the core protein in the binding of FGF7 and PRELP stabilizing the binding.
The LG2 region of core protein C-terminus of agrin is responsible of the binding with α-dystroglycan, a component of dystrophin glycoprotein complex, [Bezakova et al., 2003] that cross the plasma membrane and become a structural link between cell cytoskeleton and extracellular matrix binding laminin. In the basal lamina of the brain microvasculature and in the muscle basement membranes, agrin core protein binds the laminin 1, 2 and 4 throughout its N-terminal domain [Bezakova et al., 2003; Iozzo, 1998; Jury and Kabouridis, 2010]. Agrin is present in two
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different isoforms, the secreted form and the transmembrane one, that takes a non cleaved signal peptide. While the secreted agrin isoform is able to bind laminin, the transmembrane agrin isoform is not able to do it. The presence or not of this signal peptide influences the localization of agrin in tissues that do or do not contain a basal laminin [Bezakova et al., 2003]. Agrin is essential in motoneurons and in the brain; in the first it pays an important role in the formation of neuromuscular junctions, while in the second it is essential for the integrity of the blood brain barrier. Mutant mice deficient in agrin show non-functional NMJs and an uncontrolled immune cell infiltration in the brain throughout the BBB and they die before or shortly after birth [Jury and Kabouridis, 2010].
NG2/CSPG4 extended central D2 domain binds to type V and VI collagen, acting as a linkage between the cell surface and the extracellular matrix. Similar results have been achieved on laminin 2-coated surfaces. The roles of collagen VI and laminin 2 in brain vasculature and their association with axonal processes provide a means for migration of NG2-positive glioma cells along blood vessels and nerve fiber tracts [Couchman, 2010; Xu et al., 2011].
Also for syndecans, the core proteins is involved in integrins binding, in particular unglycosylated syndecan 1, 2 and 4 ectodomains can promote integrins-mediated adhesion. In the case of syndecan-1, a specific region approximately in the middle of the ectodomain interacts directly with β3 or β5 integrins, instead syndecan 2 and 4 ectodomains are able to activate β1 integrins in an indirect way [Couchman, 2010]. Syndecan 1 is responsible for the impaired cell spreading on vitronectin but not on fibronectin thanks to its ability to modulate vitronectin interaction with α5β3 integrin receptors [Tkachenko et al., 2005]. The region of the syndecan 1 ectodomain that interacts with integrin has been identified, it is termed synstatin and located between the heparan sulfate substitution sites and the transmembrane domain. Synstatin is so called for its ability to act as a competitor of intact proteoglycans and inhibitor of angiogenesis and tumor progression [Choi et al., 2011]. Syndecans not only bind their ligands thorough their extracellular domain, but they can influence integrins-mediated adhesion also through the intracellular signaling routes [Couchman, 2010]. In this way syndecans can modulate different integrin-dependent processes like fibronectin matrix assembly.
Chondroadherin is mainly found in the territorial matrix of articular cartilage where it binds the α2β1 integrins on the cell surface of chondrocytes and induces cells to remain round. Studies on a chondrosarcoma cell line, K9 cells, show that these cells are able to bind both native and unfolded chondroadherin, suggesting that the binding is due to a linear amino acid segment of the chondroadherin molecule [Haglund et al., 2011]. This segment is shown to be located in one of the two cysteine loops in the C terminus of chondroadherin [Haglund et al., 2011]. The binding induces
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ERK phosphorylation in human articular cartilage chondrocytes, mediating signals between the chondrocytes and the cartilage matrix. Chondroadherin also interact with collagen type II influencing the collagen fibrillogenesis [Haglund et al., 2011]. It is demonstrated that complexes of monomeric collagen type II and chondroadherin can be released from articular cartilage to activate resident matrix metalloproteinases. Both chondroadherin and collagen interact with chondrocytes, partly via the same receptor, but give rise to different cellular responses. By also interacting with each other, a complex system is created which may be of functional importance for the communication between the cells and its surrounding matrix and/or in the regulation of collagen fibril assembly.
PRELP is present in or close to several basement membranes. This structure is found as a thin sheet of extracellular matrix separating epithelial cells from the underlying connective tissue.
PRELP binds the HS chains of perlecan and bridges the collagens type I and II fibres of matrix with these HSPGs at the cell surface, showing an important role in cell-matrix adhesion [Happonen et al., 2011; Rucci et al., 2009]. This binding occurs via N-terminal region of PRELP, because full-length PRELP, but not truncated PRELP lacking the amino-terminal domain, bound perlecan, while the bind with collagen occurs via its LRR domain and truncation of PRELP had not disturbed the protein conformation. By binding to perlecan HS chains in the basement membrane via its amino-terminal part and to collagen in the connective tissue via its LRR domain, PRELP is a likely candidate as one of the anchoring molecules at basement membrane-connective tissue junctions.
Osteoadherin/osteomodulin was firstly extracted from bovine cartilage and then it was discovered also in human cartilage and bone matrices, where it seems to be osteoblasts-specific. It binds to hydroxyapatite and its potential function is to bind cells, since it is showed to be as efficient as fibronectin in promoting osteoblasts attachment in vitro. Its involvement in osteoblasts attachment is due to its interaction with α5β1 integrin [Bleicher et al., 2000]. In vitro experiments on rat osteosarcoma cells suggest that the integrins-binding is due to the core protein RGD region of osteoadherin. Moreover in vitro experiments on MC3T3E1 osteoblasts show that osteoadherin overexpression in these cells increase the cell differentiation and mineralization and reduces their proliferation and migration. Probably this anti-motility function may berelated with the osteoadherin capacity to bind and blocking EGFR. EGFR deficient mice demonstrate a reduced osteoblasts migration and proliferation and EGFR expression is upregulated in bone metastatic cancers. LRR domain of the OSD core protein is indicated as the EGFR-binding site.
32 ONLY GAG CHAINS
In some cases GAG chains binding capacity may play a pivotal role in cell-matrix adhesion.
In particular cel surface syndecans may interact with different ECM molecules like fibronectin, vitronectin, laminin, collagens, plasma protein and antithrombin 1 and with different growth factors and morphogens. The binding is due to their GAG chains that are able to recognizes and binds the heparin-binding sites of these molecules. More deeply, in myeloma cells syndecan 1 binds tenascin with different affinities depending on its degree of glycosylation and modification of its GAG chains [Choi et al., 2011]. In focal adhesions both integrins and proteoglycans are present together and it is well-known that focal adhesion formation during cell spreading on fibronectin depends upon engagement of an integrin and a cell-surface proteoglycan [Choi et al., 2011]. So the cooperation between integrins and syndecans became really crucial for cell adhesion to different matrices. In particular syndecan 1-mediated signaling promotes cell spreading in human mammary carcinoma cells, a process that requires cooperation with α5β3 integrin, and it supports α2β1 integrin-mediated adhesion on collagen type I and type II. It is shown that syndecan 1 and β1 integrin cooperatively regulate adhesion and MMPs production by human salivary gland tumors on the laminin α1-derived peptide AG73 [Choi et al., 2011]. Syndecan 2 cooperates with α5β1 integrin in cell adhesion to fibronectin and regulates actin cytoskeletal organization in Lewis lung carcinoma cells. In human colon cancer cells, syndecan 2 regulates adhesion and migration through cooperation with α2β1 integrin. Moreover syndecan 4 binds through its GAG chains to the heparin-binding domain of fibronectin and promotes the formation of focal adhesions and stress fibers.
Some studies suggest that CS chains may cooperate with HSs in binding to extracellular matrix protein laminin but they have not a principle role in the binding [Tkachenko et al., 2005].
Fibronectin interactions with heparan sulfate that mediate proteoglycan-based cell adhesion both in vitro and in vivo require N-sulfation, but not 2-O-sulfation, of the chains [Choi et al., 2011]. More deeply, integrin α5β1, but not α4β1 integrin or its close relative α9β1, requires syndecan 4 as a co-receptor and can signal to induce focal adhesion formation and migration by increasing PKCα activation. Syndecan 4 is involved also in the regulation of astrocyte adhesion through a cooperative interaction with α5β3 integrin, and clustering of syndecan 4 and β1 integrin by laminin α3 chain-derived peptide promotes keratinocyte migration [Choi et al., 2011]. RNAi knockdown and mutagenesis studies have subsequently revealed cooperation of α5β3 integrin and α5β5 integrin with syndecan 1 during adhesion to vitronectin 7, 8, and α2β1 integrin and α6β4 integrin with syndecans during adhesion to laminin [Morgan et al., 2007]. So the role of the complexes syndecan-integrin became really foundamental in the process of matrix assembly.
33 COOPERATION
The cooperation of GAG chains and core protein is the basis of endocan functions. In particular it binds fibronectin, collagens and cytokines via its GAG chain. Instead the EGF-like domain of core protein is involved in the binding of integrins, in particular of α5β3 integrins, a cell surface receptor that is present on the apical side of endothelial cells. This bind occurs in the presence of divalent cations. So endocan may cooperate with integrins to promote focal adhesion complexes assembly and disassembly influencing cell adhesion and migration that are really important during the endothelial-mesencymal transition [Carrillo et al., 2011].